1. Field of the Invention
Embodiments of the invention relate to Vitamin B12 conjugates for oral delivery of proteins and peptides, and more particularly, to Vitamin B12 conjugated to insulin. Methods of preparing and using such conjugates in treatment of disease, particularly diabetes are described.
2. Description of the Related Art
Oral, enteric delivery of insulin is potentially an attractive means for non-invasive insulin delivery since it is likely to have high patient compliance. Previous research has demonstrated some promising results using the oral-enteric route but the bioavailability of only about 5% is low. Two major limitations related to successful oral-enteric delivery are proteolysis in the gastrointestinal tract and poor absorption from the intestine into the blood (Heinemann, L., et al., Current Pharmaceutical Design 2001, 7(14), 1327-1351; Shah, R. B., et al., Critical Reviews in Therapeutic Drug Carrier Systems 2002, 19(2), 135-169).
Specific uptake mechanisms exist in the gastrointestinal tract for uptake of dietary molecules. In the case of Vitamin B12, a specific binding protein is released into the intestine which binds to its ligand in the lumen of the gut. Mammals have a transport mechanism for the absorption and cellular uptake of the relatively large Vitamin B12 molecule which relies upon complexing to a naturally occurring transport protein known as Intrinsic Factor (Chemistry and Biochemistry of B12, Chapters 16 (Intrinsic Factor, Haptocorrin and their receptors) and 17 (Transcobalamin II), Banerjee, Ruma (Ed), Wiley Interscience 1999; Vitamin B12 Zagalak, et al., (Eds), de Gruyter Press 1979). Russell-Jones et al. (U.S. Pat. Nos. 5,428,023 & 5,807,832) have shown that Vitamin B12 can be coupled to a peptide, e.g., the D-Lys-6-analog of luteinizing hormone releasing hormone, so as to preserve the ability of Vitamin B12 to interact with Intrinsic Factor, and thereby take advantage of the natural uptake mechanism for Vitamin B12 to deliver the luteinizing hormone releasing hormone analog into the blood. Russell-Jones et al. teach attachment of the luteinizing hormone releasing hormone analog to Vitamin B12 at a carboxyl group of an acid-hydrolyzed propionamide side chain (see FIG. 1).
Other proteins and peptides have also been conjugated to Vitamin B12 in attempts to provide effective oral delivery compositions. For example, U.S. Pat. No. 5,574,018 teaches Vitamin B12 conjugated to erythropoietin, granulocyte colony stimulating factor and consensus interferon through covalent binding at the primary hydroxyl site of the ribose moiety of the Vitamin B12. Conjugates of other bioactive agents and Vitamin B12 are taught by Grissom et al. (WO 01/30967 & WO 98/08859). Grissom et al. teach covalent attachment of cancer treatment drugs to the cobalt atom of Vitamin B12. In some cases, see e.g., U.S. Pat. No. 6,482,413, the Vitamin B12 is not directly linked to the target peptide or protein, but rather the Vitamin B12 is linked to micro or nanocapsules containing unconjugated, intact peptide or protein. Although this approach is touted by the patentee as providing better protection against proteolysis and Vitamin B12-mediated transport of larger payloads of biologically active peptide or protein, it presents many more technical issues related to polymer encapsulation technology and inefficient transport of the relatively large particles across the intestinal lining.
Despite the theoretical advantages of using a conjugate of Vitamin B12 and insulin to provide an oral delivery form of insulin, no one has been successful in developing an effective conjugate. Indeed, researchers have concluded that it is not possible to link insulin to Vitamin B12 such that the resulting conjugate is capable of ushering a therapeutically effective amount of biologically active insulin across the intestinal lining (see e.g., Table 1, Russell-Jones, G. J. 1998 Crit. Rev. Ther. Drug Carrier Syst. 15:557-586, indicating that Vitamin B12 alone lacks the necessary capacity to transport insulin from the intestine into the blood).
Accordingly, there remains an important and unmet need for an oral delivery form of insulin, wherein adequate levels of active insulin are deliverable into the blood from the intestine using the Vitamin B12-Intrinsic Factor uptake mechanism.